CN112939868B - Indazole hydrazide compound and application thereof - Google Patents
Indazole hydrazide compound and application thereof Download PDFInfo
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Abstract
The invention provides an indazole hydrazide compound shown as a formula (I); wherein R is selected from substituted alkyl, substituted alkenyl or substituted phenyl; said substituted alkyl andthe substituents in the substituted alkenyl group include phenyl and/or substituted phenyl; r' is selected from H or alkyl. Compared with the prior art, the indole hydrazide compound provided by the invention can be used as an integrin av beta 3 receptor antagonist, has obvious activity of resisting prostate cancer, and has a significant inhibiting effect on an enzalutamide-resistant cell line.
Description
Technical Field
The invention belongs to the technical field of drug synthesis, and particularly relates to an indazole hydrazide compound and application thereof.
Background
Prostate cancer is one of the most common malignant tumors in men in China and Western countries, the incidence rate of the prostate cancer rapidly rises with the continuous aging of the population in China and the change of living habits, and two thirds of prostate cancer patients lose the chance of radical treatment after being diagnosed in an advanced stage. Endocrine therapy based on antiandrogens is the first treatment option for advanced prostate cancer, and initially satisfactory efficacy is obtained, but after 12 to 18 months, most patients develop Castration Resistant Prostate Cancer (CRPC) with high metastasis, with 90% of metastatic CRPC occurring in the skeleton.
For CRPC patients with drug resistance of the first generation antiandrogen drugs, the clinical recommendation is to use novel antiandrogen drugs such as abiraterone and enzalutamide. Although abiraterone and enzalutamide can prolong the survival period of CRPC patients, clinical practice and large-scale clinical trial studies show that the novel antiandrogen drug can not avoid the generation of primary or acquired drug resistance.
The interaction of the extracellular matrix with tumor cells is a driver of the development of CRPC resistance. The interaction between tumor cells and the Extracellular matrix (ECM) promotes tumor cell proliferation, invasion and metastasis. Recent studies have shown that ECM-mediated drug resistance is an important factor affecting tumor progression and therapeutic response. In bone metastasis, CRPC, many integrin families, TGF- β families, bone-resident proteins, RANKL and PTHrP, etc. are involved in matrix remodeling and affect prostate cancer resistance. Therefore, the development of drugs targeting the interaction of tumor cells and microenvironment matrices is expected to be a new therapeutic strategy against CRPC resistance.
Integrin receptors are important extracellular matrices affecting tumor metastasis and drug resistance. Wherein, the alpha v beta 3 receptor is expressed on tumor cells and host matrix cells, blocks the interaction between extracellular matrix and integrin receptor alpha v beta 3, and can play the role of resisting tumor and metastasis. Cilengitide is an integrin receptor inhibitor of av β 3 and several phase II clinical trials have been conducted in different types of tumors, including castration-resistant prostate cancer. Although the monotherapy with cilengitide is well tolerated, the antitumor activity is relatively weak and eventually the clinical trial of cilengitide phase 3 ends up failing.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide an indazole hydrazide compound having a good inhibitory effect on integrin av β 3 receptor and an application thereof.
The invention provides an indazole hydrazide compound shown as a formula (I):
wherein R is selected from substituted alkyl, substituted alkenyl or substituted phenyl;
the substituent in the substituted alkyl and the substituted alkenyl comprises phenyl and/or substituted phenyl;
r' is selected from H or alkyl.
Preferably, R is selected from substituted C2-C10 alkyl, substituted C2-C10 alkenyl or substituted phenyl;
r' is selected from H or C1-C10 alkyl.
Preferably, the substituent in the substituted phenyl is selected from one or more of C1-C5 alkyl, C1-C5 alkenyl, hydroxyl, C1-C5 alkoxy, nitro and C1-C5 haloalkyl.
Preferably, the indazole hydrazide compound is selected from one of the formulas (I-1) to (I-5):
wherein n and m are each independently an integer of 1 to 5;
R 1 ～R 8 each independently selected from C1-C5 alkyl, C1-C5 alkenyl, hydroxyl, C1-C5 alkoxy, nitro or C1-C5 halogenated alkyl;
r' is selected from H or C1-C5 alkyl.
Preferably, n and m are each independently an integer of 1 to 3;
R 1 ～R 8 each independently selected from C1-C3 alkyl, C1-C3 alkenyl, hydroxyl, C1-C3 alkoxy, nitro or C1-C3 halogenated alkyl;
r' is selected from H or C1-C3 alkyl.
Preferably, the indazole hydrazide compound is selected from one of formulae (1) to (12):
the invention also provides application of the indazole hydrazide compound as an integrin av beta 3 receptor antagonist.
The invention also provides application of the indazole hydrazide compound in preparation of tumor drugs.
Preferably, the tumor is one or more of prostate cancer, melanoma, and ovarian cancer.
Preferably, the tumor is resistant to enzalutamide treatment.
The invention provides an indazole hydrazide compound shown as a formula (I); wherein R is selected from substituted alkyl, substituted alkenyl or substituted phenyl; the substituent in the substituted alkyl and the substituted alkenyl comprises phenyl and/or substituted phenyl; r' is selected from H or alkyl. Compared with the prior art, the indole hydrazide compound provided by the invention can be used as an integrin av beta 3 receptor antagonist, has obvious activity of resisting prostate cancer, and has a significant inhibiting effect on an enzalutamide-resistant cell line.
Experiments show that the KD value of the interaction between the indazole hydrazide compound shown in the formula (5) and the protein alpha v beta 3 is 158nM; the indazole hydrazide compound shown in the formula (5) is effective on various tumor cells, including prostatic cancer, melanoma, ovarian cancer and the like, and also has an inhibitory effect on vascular endothelial cells. And combined drug research is carried out on the prostatic cancer enzalutamide-resistant 22RV1 cells, and the indazole hydrazide compound shown in the formula (5) and enzalutamide have synergistic effect.
Drawings
FIG. 1 is a NMR chart of a compound represented by the formula (1) in example 1 of the present invention;
FIG. 2 is a nuclear magnetic resonance carbon spectrum of a compound represented by the formula (1) in example 1 of the present invention;
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by the formula (3) in example 1 of the present invention;
FIG. 4 is a nuclear magnetic resonance carbon spectrum of a compound represented by the formula (3) in example 1 of the present invention;
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by the formula (4) in example 1 of the present invention;
FIG. 6 is a nuclear magnetic resonance carbon spectrum of a compound represented by the formula (4) in example 1 of the present invention;
FIG. 7 is a NMR spectrum of a compound represented by the formula (5) in example 1 of the present invention;
FIG. 8 is a NMR spectrum of a compound represented by the formula (5) in example 1 of the present invention;
FIG. 9 is a NMR spectrum of a compound represented by the formula (6) in example 1 of the present invention;
FIG. 10 is a nuclear magnetic resonance carbon spectrum of a compound represented by the formula (6) in example 1 of the present invention;
FIG. 11 is a NMR hydrogen spectrum of a compound represented by the formula (7) in example 1 of the present invention;
FIG. 12 is a NMR spectrum of a compound represented by the formula (7) in example 1 of the present invention;
FIG. 13 is a NMR spectrum of a compound represented by the formula (8) in example 1 of the present invention;
FIG. 14 is a NMR spectrum of a compound represented by the formula (8) in example 1 of the present invention;
FIG. 15 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by the formula (9) in example 1 of the present invention;
FIG. 16 is a NMR carbon spectrum of a compound represented by the formula (9) in example 1 of the present invention;
FIG. 17 is a NMR spectrum of a compound represented by the formula (10) in example 1 of the present invention;
FIG. 18 is a NMR carbon spectrum of a compound represented by the formula (10) in example 1 of the present invention;
FIG. 19 is a schematic representation of the formulation and dilution of an indazole hydrazide compound of example 2 of the present invention;
FIG. 20 is a schematic view of sample addition of a drug to be tested in embodiment 2 of the present invention;
FIG. 21 is a graph showing the activity of the compound represented by the formula (5) in example 2 of the present invention on various tumor cells;
FIG. 22 is a graph showing the activity of the compound represented by the formula (5) in example 2 of the present invention on various tumor cells;
FIG. 23 is a graph of a fitted pharmacodynamic profile using chemiluminescence to determine the administration of a compound of formula (5) in combination with enzalutamide to cell 22RV 1;
FIG. 24 is a graph showing the activity of the compound represented by the formula (1) (code No. 19-1), the compound represented by the formula (3) (code No. 19-2), the compound represented by the formula (2) (code No. 19-3), the compound represented by the formula (4) (code No. 19-4) and the compound represented by the formula (10) (code No. 19-5) against tumor cells 22RV 1;
FIG. 25 is a graph showing the activity of the compound represented by the formula (7) (code 19-6), the compound represented by the formula (8) (code 19-7), the compound represented by the formula (6) (code 19-8) and the compound represented by the formula (9) (code 19-9) against tumor cells 22RV 1;
FIG. 26 is a graph showing the activity of the compound represented by the formula (1) (code 19-1), the compound represented by the formula (3) (code 19-2), the compound represented by the formula (2) (code 19-3), the compound represented by the formula (4) (code 19-4) and the compound represented by the formula (10) (code 19-5) against tumor cells LNCaP;
FIG. 27 is a graph showing the activity of the compound represented by the formula (7) (code 19-6), the compound represented by the formula (8) (code 19-7), the compound represented by the formula (6) (code 19-8) and the compound represented by the formula (9) (code 19-9) against tumor cells LNCaP.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The invention provides an indazole hydrazide compound, which is shown as a formula (I):
wherein R is selected from substituted alkyl, substituted alkenyl or substituted phenyl, preferably substituted C2-C10 alkyl, substituted C2-C10 alkenyl or substituted phenyl, more preferably substituted C2-C8 alkyl, substituted C2-C8 alkenyl or substituted phenyl, and still more preferably substituted C2-C6 alkyl, substituted C2-C6 alkenyl or substituted phenyl; the substituents in the substituted alkyl and substituted alkenyl groups include phenyl and/or substituted phenyl.
R' is H or alkyl, preferably H or C1-C10 alkyl, more preferably H or C1-C5 alkyl, even more preferably H or C1-C3 alkyl, and most preferably H or methyl.
According to the invention, the substituent group in the substituted phenyl is preferably one or more of C1-C5 alkyl, C1-C5 alkenyl, hydroxyl, C1-C5 alkoxy, nitro and C1-C5 haloalkyl, more preferably one or more of C1-C3 alkyl, C1-C3 alkenyl, hydroxyl, C1-C3 alkoxy, nitro and C1-C3 haloalkyl, and still more preferably one or more of C1-C2 alkyl, C1-C2 alkenyl, hydroxyl, C1-C2 alkoxy, nitro and C1-C2 haloalkyl; the halogen atom in the haloalkyl group is preferably fluorine, chlorine, bromine or iodine, more preferably fluorine; the number of halogen atoms in the haloalkyl group is preferably 1 to 3.
In the invention, the indazole hydrazide compound is further specifically selected from one of the formulas (I-1) to (I-5):
wherein n and m are each independently an integer of 1 to 5, more preferably an integer of 1 to 4, still more preferably an integer of 1 to 3, and most preferably 1 or 2.
R 1 ～R 8 Each independently is preferably C1-C5 alkyl, C1-C5 alkenyl, hydroxyl, C1-C5 alkoxy, nitro or C1-C5 haloalkyl, more preferably one or more of C1-C3 alkyl, C1-C3 alkenyl, hydroxyl, C1-C3 alkoxy, nitro and C1-C3 haloalkyl, and still more preferably one or more of C1-C2 alkyl, C1-C2 alkenyl, hydroxyl, C1-C2 alkoxy, nitro and C1-C2 haloalkyl; the halogen atom in the haloalkyl group is preferably fluorine, chlorine, bromine or iodine, more preferably fluorine; the number of halogen atoms in the haloalkyl group is preferably 1 to 3.
Most preferably, the indazole hydrazide-type compound is selected from one of formulae (1) to (12):
the indole hydrazide compound provided by the invention can be used as an integrin av beta 3 receptor antagonist, has obvious activity of resisting prostate cancer, and has a significant inhibiting effect on an enzalutamide-resistant cell line.
The invention also provides a preparation method of the indazole hydrazide compound, which comprises the following steps: reacting the compound shown in the formula (II) with RCOR' to obtain the compound shown in the formula (I).
Reacting the compound represented by the formula (II) with RCOR', preferably in a solvent; the solvent is preferably an alcoholic solvent, more preferably ethanol; the reaction is preferably a reflux reaction; the molar ratio of the compound of formula (II) to RCOR' is preferably 1: (1-1.2); the embodiment provided by the invention is specifically 1:1.1.
the compound represented by the formula (II) is preferably carried out according to the following steps: reacting the compound shown in the formula (III) with hydrazine to obtain the compound shown in the formula (II).
Wherein R "is a C1-C5 alkyl group, more preferably a C1-C4 alkyl group, and still more preferably an ethyl group.
Reacting the compound shown in the formula (III) with hydrazine preferably in a solvent; the solvent is preferably an alcoholic solvent, more preferably ethanol; the molar ratio of the compound represented by formula (III) to hydrazine is preferably 1: (6 to 8), more preferably 1:7; the reaction is preferably a reflux reaction; the reaction time is preferably 20 to 30 hours; after the reaction, the solvent was removed, and the reaction mixture was washed with ethyl acetate and water to obtain a compound represented by the formula (II).
The compound represented by the formula (III) is preferably prepared according to the following steps: heating the compound shown in the formula (IV) and hydrazine in acetic acid for reaction to obtain the compound shown in the formula (III).
The molar ratio of the compound represented by the formula (IV) to hydrazine is preferably 1: (1 to 1.2), more preferably 1: (1.1-1.2); the heating reaction is preferably a reflux reaction; the heating reaction time is preferably 40-70 min, and more preferably 60min; after the heating reaction is finished, preferably pouring into ice water, neutralizing with sodium bicarbonate, extracting with ethyl acetate, concentrating the extract, and purifying by column chromatography with a mixed solution of n-hexane and ethyl acetate to obtain a compound shown in formula (III); the volume ratio of the n-hexane to the ethyl acetate is preferably 3:1.
the compound represented by the formula (IV) is preferably prepared according to the following steps: reacting cyclohexanone with (COOR') in sodium ethoxide solution, decomposing the reaction mixture with sulfuric acid solution, extracting with ethyl acetate, concentrating the extract, and purifying with column chromatography with mixed solution of n-hexane and ethyl acetate to obtain compound shown in formula (IV); the reaction time is preferably 10 to 14 hours, and more preferably 12 hours; the volume of the n-hexane and the ethyl acetate is preferably 12:1.
the invention also provides application of the indazole hydrazide compound as an integrin av beta 3 receptor antagonist.
The invention also provides application of the indazole hydrazide compound in preparation of tumor drugs.
Wherein, the tumor is preferably one or more of prostate cancer, melanoma and ovarian cancer.
Further specifically, the tumor is preferably a tumor resistant to enzalutamide treatment.
Further specifically, the invention provides application of the indazole hydrazide compound in preparation of medicines for inhibiting tumor cells 22RV1, 22RV1-SPP1 and LNCaP.
In order to further illustrate the present invention, the indazole hydrazide compound and the application thereof provided by the present invention are described in detail in the following examples.
The reagents used in the following examples are all commercially available.
Example 1
The compounds represented by the formulae (1), (2) to (10) were prepared according to the following procedures:
sodium ethoxide solution was prepared by adding sodium (1.5g, 65mmol) to absolute ethanol (20 mL) at 0 ℃; then a mixture of cyclohexanone (4.41g, 44mmol) and diethyl oxalate (7.3g, 50mmol) was slowly added and the solution was stirred at room temperature for 12h, after decomposing the reaction mixture with 2N sulfuric acid solution, the mixture was extracted with ethyl acetate, dried and concentrated in organic solvent to give crude product which was purified with N-hexane: further purification by column chromatography of ethyl acetate (12.
Hydrazine (448mg, 14mmol) was slowly added to a cooled suspension of product 2 (2.38g, 12mmol) in acetic acid (5 mL), the mixture was heated at reflux for 1h, poured into ice water and quenched with NaHCO 3 Neutralized and extracted with ethyl acetate, the organic phases were combined and dried (Na) 2 SO 4 ) Filtered and concentrated, then treated with n-hexane: the residue was purified by column chromatography on ethyl acetate (3:1) to give product 3 as a white solid (2g, 86%).
Product 3 (970 mg, 5mmol) and hydrazine (1.34g, 35mmol) were heated to reflux in ethanol (10 mL) for 1 day. After the reflux was complete, the ethanol was evaporated and the precipitate was collected by filtration, washed with ethyl acetate and water to give hydrazide 4 (580 mg, 64%) as a white solid.
Hydrazide 4 (72mg, 0.4 mmol) and substituted aldehyde RCOR' (0.44 mmol) were refluxed in ethanol (3 mL) to obtain precipitate compound 5, which was then collected by filtration and washed with cold ethanol (81%).
The obtained compounds represented by formula (1), formula (3) to formula (10) were analyzed by nuclear magnetic resonance to obtain spectra shown in fig. 1 to 18; wherein FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by the formula (1); FIG. 2 is a nuclear magnetic resonance carbon spectrum of a compound represented by the formula (1); FIG. 3 is a NMR chart of a compound represented by the formula (3); FIG. 4 is a nuclear magnetic resonance carbon spectrum of the compound represented by the formula (3); FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by the formula (4); FIG. 6 is a nuclear magnetic resonance carbon spectrum of the compound represented by the formula (4); FIG. 7 is a NMR hydrogen spectrum of a compound represented by the formula (5); FIG. 8 is a nuclear magnetic resonance carbon spectrum of a compound represented by formula (5); FIG. 9 is a nuclear magnetic resonance hydrogen spectrum of the compound represented by formula (6); FIG. 10 is a nuclear magnetic resonance carbon spectrum of a compound represented by the formula (6); FIG. 11 is a NMR hydrogen spectrum of a compound represented by the formula (7); FIG. 12 is a NMR carbon spectrum of a compound represented by the formula (7); FIG. 13 is a nuclear magnetic resonance hydrogen spectrum of the compound represented by the formula (8); FIG. 14 is a nuclear magnetic resonance carbon spectrum of the compound represented by the formula (8); FIG. 15 is a nuclear magnetic resonance hydrogen spectrum of a compound represented by the formula (9); FIG. 16 is a nuclear magnetic resonance carbon spectrum of the compound represented by the formula (9); FIG. 17 is a nuclear magnetic resonance hydrogen spectrum of the compound represented by formula (10); FIG. 18 is a nuclear magnetic resonance carbon spectrum of the compound represented by formula (10).
The following nuclear magnetic resonance results were obtained:
an indazole hydrazide compound represented by the formula (1):
1 H NMR(400MHz,DMSO-d 6 )δ12.84(s,1H),9.77(s,1H),9.16(s,1H),7.03(d,J＝8.3Hz,2H),6.66(d,J＝8.4Hz,2H),2.75–2.56(m,6H),2.50–2.45(m,2H),1.91(s,3H),1.76–1.60(m,4H).
13 C NMR(101MHz,DMSO-d 6 )δ158.54,157.05,155.46,141.29,140.16,131.42,129.22,116.34,115.08,40.77,31.28,22.62,21.98,20.97,20.60,15.55.
an indazole hydrazide compound represented by the formula (3):
1 H NMR(400MHz,DMSO-d 6 )δ12.86(s,1H),11.16(s,1H),8.31(s,1H),7.43–7.28(m,3H),7.17–7.12(m,2H),6.07(t,J＝7.6Hz,1H),2.57(d,J＝6.0Hz,4H),1.95(t,J＝7.2Hz,2H),1.76–1.58(m,5H),0.82(d,J＝6.6Hz,6H).
13 C NMR(101MHz,DMSO-d 6 )δ158.89,151.63,141.23,139.95,139.65,139.57,135.93,129.64,127.86,127.03,116.55,37.79,28.13,22.60,22.33,21.96,20.93,20.56.
an indazole hydrazide compound represented by the formula (4):
1 H NMR(400MHz,DMSO-d 6 )δ12.89(s,1H),11.41(s,1H),8.45(s,1H),7.55(d,J＝7.8Hz,2H),7.25(d,J＝7.8Hz,2H),2.65(dt,J＝25.1,6.2Hz,4H),2.33(s,3H),1.78–1.62(m,4H).
13 C NMR(101MHz,DMSO-d 6 )δ159.10,146.65,141.19,139.95,139.39,131.99,129.34,126.79,116.62,22.58,21.94,20.96,20.96,20.54.
an indazole hydrazide compound represented by the formula (5):
1 H NMR(400MHz,DMSO-d 6 )δ12.92(s,1H),11.46(s,1H),9.62(s,1H),8.38(s,1H),7.23(t,J＝7.8Hz,1H),7.13(s,1H),7.03(d,J＝7.5Hz,1H),6.80(dd,J＝8.1,2.4Hz,1H),2.64(dt,J＝21.7,6.1Hz,4H),1.71(dd,J＝13.7,6.6Hz,4H).
13 C NMR(101MHz,DMSO-d 6 )δ159.17,157.65,146.77,141.18,140.06,136.01,129.86,118.55,117.09,116.73,112.50,22.63,21.99,21.05,20.59.
an indazole hydrazide compound represented by the formula (6):
1 H NMR(400MHz,DMSO-d 6 )δ13.00(s,1H),11.89(s,1H),8.59(s,1H),8.29(d,J＝8.6Hz,2H),7.91(d,J＝8.5Hz,2H),2.65(dt,J＝22.2,6.2Hz,4H),1.70(dd,J＝13.7,6.6Hz,4H).
13 C NMR(101MHz,DMSO-d 6 )δ159.41,147.61,144.16,141.14,140.92,140.23,127.74,124.14,117.05,22.60,21.97,21.03,20.58.
an indazole hydrazide compound represented by the formula (7):
1 H NMR(400MHz,DMSO-d 6 )δ12.91(s,1H),11.39(s,1H),9.57(s,1H),8.35(s,1H),7.20(s,1H),7.11(d,J＝7.7Hz,1H),6.92(d,J＝7.5Hz,1H),2.64(dt,J＝22.6,6.2Hz,4H),2.13(s,3H),1.71(dd,J＝13.4,6.8Hz,4H).
13 C NMR(101MHz,DMSO-d 6 )δ159.13,155.70,146.97,141.27,140.05,133.47,130.85,126.44,118.89,116.68,111.42,22.66,22.02,21.08,20.62,16.11.
an indazole hydrazide compound represented by the formula (8):
1 H NMR(400MHz,DMSO-d 6 )δ12.91(s,1H),11.41(s,1H),8.39(s,1H),7.29(s,1H),7.11(d,J＝8.2Hz,1H),7.01(d,J＝8.3Hz,1H),3.81(s,3H),3.79(s,3H),2.64(dt,J＝23.9,6.2Hz,4H),1.71(dq,J＝12.6,6.5,5.0Hz,4H).
13 C NMR(101MHz,DMSO-d 6 )δ159.13,150.48,149.05,146.83,141.29,140.05,127.47,121.61,116.68,111.45,107.96,55.57,55.41,22.66,22.02,21.05,20.62.
an indazole hydrazide compound represented by the formula (9):
1 H NMR(400MHz,DMSO-d 6 )δ12.88(s,1H),11.30(s,1H),9.28(s,1H),8.32(s,1H),7.21(s,1H),7.02–6.88(m,2H),3.79(s,3H),2.70–2.56(m,4H),1.80–1.61(m,4H).
13 C NMR(101MHz,DMSO-d 6 )δ159.02,149.49,146.85,146.82,141.30,139.97,127.57,119.91,116.58,112.26,111.86,55.56,22.64,21.99,21.04,20.59.
an indazole hydrazide compound represented by the formula (10):
1 H NMR(400MHz,DMSO-d 6 )δ12.91(s,1H),11.72(s,1H),11.10(s,2H),9.74(s,1H),8.80(s,1H),5.81(s,2H),2.63(dt,J＝21.7,6.2Hz,4H),1.85–1.52(m,4H).
13 C NMR(101MHz,DMSO-d 6 )δ161.11,159.60,158.67,145.98,140.93,139.99,116.70,99.36,94.30,56.08,22.67,22.02,21.01,20.60,18.62.
example 3: detection of antitumor Activity
3.1 Experimental methods
3.1.1 tumor cell recovery culture
Frozen tumor cells (cells of prostate cancer, ovarian cancer and the like) are taken out from liquid nitrogen, are quickly dissolved at 37 ℃, are added into 5ml of corresponding culture medium containing FBS, are centrifuged for 5 minutes at 300g, are collected and precipitated, are fully mixed after being resuspended in the corresponding culture medium, are added into a 24-hole cell culture dish, are supplemented with the culture medium until the total volume of each hole is 2mL and the temperature is about 90 percent of confluence, and are subjected to subculture amplification or counting plating.
3.1.2 tumor cell inoculation
When the tumor cells are cultured to be passable, the cells are digested by pancreatin to prepare single cell suspension. After counting the cells, corresponding culture is performedNutrients adjusted cell concentrations to 1.2X 10 4 cells/ml, adding 90. Mu.l of the mixed solution to a 96-well plate, incubating at 37 ℃ for 30 minutes, adding the corresponding culture, culturing overnight in a 37 ℃ cell culture box, and observing the morphology and the confluence degree of tumor cells.
3.1.3 Compound formulation and dilution
After overnight tumor cell culture, compound gradient dilutions were prepared and added to the culture system.
A stock solution (100 mM) of indazole hydrazide compound dissolved in DMSO was dissolved well at room temperature, and if there were insoluble, was heated ultrasonically for 5 minutes and observed again until dissolved.
Respectively adding 20 mu l DMSO into No. 2-9 holes in a 96-hole V-shaped compound dilution plate, adding 30 mu l stock solution into 1 hole, placing the compound dilution plate on a vibration mixer, transferring 10 mu l liquid from the No. 1 hole, adding the liquid into the No. 2 hole, blowing, vibrating, mixing uniformly, transferring 10 mu l liquid from the No. 2 hole to the No. 3 hole, vibrating, blowing, mixing uniformly, sequentially repeating … until transferring 10 mu l liquid from the No. 8 hole to the No. 9 hole, blowing, vibrating, mixing uniformly, and preparing 1000X gradient diluted stock solution. [ note: DMSO readily absorbed moisture from the air, and after handling, the compound dilution plates were immediately sealed with a sealing film, stored at 4 degrees celsius, and discarded after 1 week. When the moisture absorption type sealing film is used again, the sealing film is opened after the temperature reaches the room temperature, and the concentration inaccuracy caused by moisture absorption is avoided. "C (B)
Taking a 96-hole sterile cell culture plate, transferring 2 mu l of stock solution to 1-9 holes corresponding to the No. 1-9 holes of the 96-hole sterile cell culture plate from 1000X stock solution by using a microsyringe in Kong Jiaru mu l of culture medium of No. 1-9 in an ultra-clean workbench, shaking, blowing, beating and uniformly mixing to prepare 10X stock solution. The stock solution needs to be prepared and used on the same day.
10 μ l of the 10 Xstock solution prepared on the day was added to the cell culture medium.
Referring to fig. 19, fig. 19 is a schematic of indazole hydrazide compound formulation and dilution.
3.1.4 adding the drug to be tested
After observing that the adherent growth of the tumor cells is good, 10. Mu.L of the compound with the corresponding concentration is added according to the Plate map, and the 10 Xstock solution is prepared on the same day. Incubate at 37 ℃ for 72h with 5% carbon dioxide.
Referring to FIG. 20, FIG. 20 is a schematic view of loading of the test drug, wherein Null is a blank well; DMSO, wells to which DMSO is added; BEZ235: 2.5. Mu.M BEZ235; 1 μ M Staurosporine; cpd # 19 is an amide compound represented by the formula (5).
3.1.5 chemiluminescent assay
And measuring the ATP level of the cells by adopting a chemiluminescence method, and further evaluating the cell viability. The specific operation is carried out according to the instruction, 50 mu L of CTG solution is added after the culture is finished, the mixture is uniformly mixed, the cracking mixture is transferred to an ELISA plate, and after 5-10 minutes, chemiluminescence data is collected in an ELISA reader. The processed data were analyzed using Excel software and the IC50 was calculated from the fitted pharmacodynamic curve of the chemiluminescent data using GraphPadPrism 7 software.
3.1.6 control and quality control
In the test, the Zfactor is used as a quality control index. The Z' factor is defined by 4 parameters: mean (μ) and standard deviation (σ) of positive control (p) and negative control (n).
The calculation formula is as follows:
Z’factor＝1-(3*(σp+σn)/|(μp-μn)|)
the negative control group is an untreated group added with a solvent (DMSO); positive controls were supplemented with 2.5. Mu.M BEZ235 or 1. Mu.M Staurosporine.
Functional assays at the general cellular level Z' factor requires >0.3; the quality control pass value Z' factor of the test is set to be >0.5.
3.2 Activity data
The 10 compounds of the invention shared 12 plates in total.
The culture plates 1 to 4 were used for the cells 22RV1, wherein the culture plate 1 was used for activity detection of the compound represented by formula (5), the compound represented by formula (1), and the compound represented by formula (3), Z '(BEZ) =0.76, Z' (Staurosporine) =0.80; the plate 2 was used for activity detection of the compound represented by the formula (2), the compound represented by the formula (4) and the compound represented by the formula (10), Z '(BEZ) =0.75, Z' (Staurosporine) =0.79; the plate 3 was used for activity detection of the compound represented by the formula (7), the compound represented by the formula (8) and the compound represented by the formula (6), Z '(BEZ) =0.75, Z' (Staurosporine) =0.79; the plate 4 was used for activity detection of the compound represented by formula (9), and Z '(BEZ) =0.82, Z' (Staurosporine) =0.85.
Among them, SPP1 (Secreted phosphoprotein 1), also called Osteopontin (OPN), is a Secreted protein of the SIBLING family, and is expressed on various cells such as tumor cells, macrophages, osteoclasts, fibroblasts, and epithelial cells. The SPP1 mainly realizes the function by combining RGD (arginine-glycine-aspartic acid) and non-RGD with cell surface integrin receptors, and the sequence SVVYGLR is adjacent to the RGD motif and is a hidden integrin binding site: it is hidden in full-length SPP1, and as the tumor progresses, SPP1 is hydrolyzed by various proteases, exposing the binding site. SPP1 plays an important role in mediating tumor cell adhesion and migration, mineralization and reconstruction of bone tissue, immunoregulation, angiogenesis, fibroblast activation and reprogramming and the like. In prostate and breast cancer, high expression of SPP1 has been shown to correlate with poor prognosis and survival of prostate cancer.
The activity profiles of the compound represented by the formula (5) on various tumor cells are shown in FIGS. 21 to 22, and-19 in FIGS. 21 and 22 is the code of the compound represented by the formula (5). As can be seen from FIGS. 21 and 22, it has significant antitumor activity against PC-3, DU145, LNCaP,22RV1 cells of prostate cancer, among which the antitumor effect against WM115, melanoma, was the highest. Also, the OVCAR-4 inhibitor has inhibitory effect on ovarian cancer cells. In addition, it also has inhibitory effect on vascular endothelial cell HUVEC-T11 and microvascular endothelial cell HMEC-1.
Fig. 23 is a graph of fitted pharmacodynamics measurements using chemiluminescence of the compound of formula (5) administered in combination with enzalutamide to cell 22RV 1. As can be seen from fig. 23, the compound represented by formula (5) in combination with enzalutamide had a synergistic effect on drug-resistant 22RV1 cells.
FIG. 24 is a graph showing the activity of the compound represented by the formula (1) (code 19-1), the compound represented by the formula (3) (code 19-2), the compound represented by the formula (2) (code 19-3), the compound represented by the formula (4) (code 19-4) and the compound represented by the formula (10) (code 19-5) against tumor cells 22RV 1.
FIG. 25 is a graph showing the activity of the compound represented by the formula (7) (code No. 19-6), the compound represented by the formula (8) (code No. 19-7), the compound represented by the formula (6) (code No. 19-8) and the compound represented by the formula (9) (code No. 19-9) against tumor cells 22RV 1.
FIG. 26 is a graph showing the activity of the compound represented by the formula (1) (code 19-1), the compound represented by the formula (3) (code 19-2), the compound represented by the formula (2) (code 19-3), the compound represented by the formula (4) (code 19-4) and the compound represented by the formula (10) (code 19-5) against tumor cells LNCaP.
FIG. 27 is a graph showing the activity of a compound represented by the formula (7) (code No. 19-6), a compound represented by the formula (8) (code No. 19-7), a compound represented by the formula (6) (code No. 19-8) and a compound represented by the formula (9) (code No. 19-9) against tumor cells LNCaP.
Table 1 shows activity data of indazole hydrazide compounds prepared in example 1 against different tumor cells.
TABLE 1 antitumor Activity data
Claims (1)
1. The combination of indazole hydrazide compounds and enzalutamide is applied to the preparation of antitumor drugs; the indazole hydrazide compound is a compound shown as a formula (5); the tumor is prostate cancer with drug resistance to enzalutamide treatment:
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